Application of a Rapid and Sensitive Method for Hormonal and Vitamin E Profiling Reveals Crucial Regulatory Mechanisms in Flower Senescence and Fruit Ripening

Knowledge of ripeness and regulation of postharvest processes is an important tool to prevent loss of commercial value in both fruit and cut flower markets. The joint analysis of hormones and vitamin E levels can reveal complex interactions between hormones and oxidative stress as key regulators of postharvest processes. Profiling of both groups of metabolic compounds was performed during the ripening of non-climacteric fruits (red raspberry, Rubus idaeus L.) and senescence of ethylene-insensitive flowers (Dutch Iris, Iris x hollandica L.). After an initial extraction of the sample, without further purification steps, the hormonal profile was analyzed by UPLC-MS/MS and vitamin E levels were measured by HPLC. This methodological approach was very fast and had enough sensitivity for the analysis of small samples. Raspberry fruit maturation was characterized by a decline of cytokinin levels [zeatin, zeatin riboside, 2-isopentenyl adenine, and isopentenyl adenosine (Z, ZR, 2-iP, and IPA, respectively)] and gibberellins (GA₁ in particular). Exogenous application of ABA prevented δ-tocopherol loss during fruit ripening. Iris floral senescence was also under strict hormonal control, also mediated by cytokinins and gibberellins. Z, ZR, 2-iP, GA₉, and GA₂₄ levels decreased in inner tepals, whereas the level of IPA decreased in style-merged-to-stigma tissues, thus suggesting tissue-specific roles for different hormones. α-Tocopherol levels decreased during senescence of inner tepals, hence suggesting enhanced oxidative stress. In conclusion, the rapid and sensitive hormonal and vitamin E profiling presented here can help in understanding the key physiological processes underlying fruit ripening and floral senescence.     

Hormonal Interactions in Fruit Development

Fruit development involves a complex interplay of cell division, differentiation and expansion of sporophytic and gametophytic tissues that is carefully coordinated temporally and spatially. Plant hormones are signal molecules that regulate many processes of plant development, including fruit development leading to mature fruit and viable mature seed. Auxins, gibberellins, cytokinins, abscisic acid, and ethylene have been implicated at various stages of fruit development. In the past, hormone application studies and hormone analysis studies have supported the hypothesis that fruit development is in part regulated by hormonal interaction. More recently, biochemical, genetic, and molecular studies are beginning to unravel the complexities of how hormones affect fruit development. In the current work, we review selected studies that show the interplay between hormones during fruit development, with an emphasis on the interaction between auxin and gibberellin in pea fruit development.     

花发育阶段以及温度对不同品种甜樱桃柱头可授性的影响

以‘布鲁克斯’、‘美早’、‘红蜜’等5个甜樱桃(Prunus avium)品种为试材,检测不同品种甜樱桃花期不同发育阶段的柱头可授性;并利用人工气候箱模拟花期低温(2 ℃/10 ℃)、常温(6 ℃/18 ℃)、高温(12 ℃/24 ℃)以及超高温(12 ℃/24 ℃/30 ℃)环境,研究不同温度处理对甜樱桃柱头可授性的影响。结果表明：(1)‘美早’、‘先锋’和‘萨米脱’适宜的授粉温度是常温(6 ℃/18 ℃)环境,‘红蜜’适宜的授粉温度是高温(12 ℃/24 ℃)环境,‘布鲁克斯’适宜授粉的温度范围更广,在常温(6 ℃/18 ℃)和高温(12 ℃/24 ℃)环境条件下其柱头均具有较高的活性。(2)‘红蜜’柱头对花期低温(2 ℃/10 ℃)和超高温(12 ℃/24 ℃/30 ℃)的抗性较强;‘布鲁克斯’和‘萨米脱’柱头对花期超高温(12 ℃/24 ℃/30 ℃)抗性较差;‘美早’柱头对花期低温(6 ℃/10 ℃)的抗性较强,但对花期高温(12 ℃/24 ℃)的抗性较差;‘先锋’柱头对花期低温(6 ℃/10 ℃)和超高温(12 ℃/24 ℃/30 ℃)均无明显抗性。研究认为,低温(2 ℃/10 ℃)对甜樱桃柱头可授性的影响主要在开花之后,而超高温(12 ℃/24 ℃/30 ℃)对整个开花过程柱头可授性的影响都很大。     

Transcriptome Analysis Reveals Candidate Lignin-Related Genes and Transcription Factors in Rosa roxburghii During Fruit Ripening

Plant Molecular Biology Reporter - The high lignin content of Rosa roxburghii fruits imparts a strong rough taste, which has restricted its use in industry. Little is known about the molecular...     

GPR88 Reveals a Discrete Function of Primary Cilia as Selective Insulators of GPCR Cross-Talk

A number of G protein-coupled receptors (GPCRs) localize to primary cilia but the functional significance of cilia to GPCR signaling remains incompletely understood. We investigated this question by focusing on the D1 dopamine receptor (D1R) and beta-2 adrenergic receptor (B2AR), closely related catecholamine receptors that signal by stimulating production of the diffusible second messenger cyclic AMP (cAMP) but differ in localization relative to cilia. D1Rs robustly concentrate on cilia of IMCD3 cells, as shown previously in other ciliated cell types, but disrupting cilia did not affect D1R surface expression or ability to mediate a concentration-dependent cAMP response. By developing a FRET-based biosensor suitable for resolving intra- from extra- ciliary cAMP changes, we found that the D1R-mediated cAMP response is not restricted to cilia and extends into the extra-ciliary cytoplasm. Conversely the B2AR, which we show here is effectively excluded from cilia, also generated a cAMP response in both ciliary and extra-ciliary compartments. We identified a distinct signaling effect of primary cilia through investigating GPR88, an orphan GPCR that is co-expressed with the D1R in brain, and which we show here is targeted to cilia similarly to the D1R. In ciliated cells, mutational activation of GPR88 strongly reduced the D1R-mediated cAMP response but did not affect the B2AR-mediated response. In marked contrast, in non-ciliated cells, GPR88 was distributed throughout the plasma membrane and inhibited the B2AR response. These results identify a discrete ‘insulating’ function of primary cilia in conferring selectivity on integrated catecholamine signaling through lateral segregation of receptors, and suggest a cellular activity of GPR88 that might underlie its effects on dopamine-dependent behaviors.     

蔬菜植物果实发育的激素调控

概述了果菜蔬菜果实中的激素来源以及激素对茄果类,瓜类,豆类蔬菜和草莓果实发育的调节作用。     

Multiplatform Molecular Profiling Reveals Epigenomic Intratumor Heterogeneity in Ependymoma

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Improvement of RT-PCR Based Detection of Two Closteroviruses Associated with Little Cherry Disease in Sweet Cherries

The increasing availability of nucleotide sequence data from plant pathogenic fruit tree viruses led recently to the development of nucleic‐acid‐based detection methods as a routine tool for diagnosis. We present a simplification and modification of the previously described test procedure for the two closteroviruses Little cherry virus 1 and 2 (LChV‐1, LChV‐2). For LChV‐1, a single tube reaction reduced unspecific amplification that led to false positive results. For the detection of LChV‐2, a primer set was developed for the local isolates of the area ‘Altes Land’ in the northern part of Germany. To determine the optimal sampling conditions for a reliable detection of both viruses different types of tissue (bark, bud, leaves) were tested in monthly intervals.     

Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker

Background

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.

Methodology and Principal Findings

Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.

Conclusion

HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.     

Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available.     

miRNA Profiling Reveals Dysregulation of RET and RET-Regulating Pathways in Hirschsprung's Disease

Hirschsprung’s disease (HSCR), the most common congenital malformation of the gut, is regulated by multiple signal transduction pathways. Several components of these pathways are important targets for microRNAs (miRNAs). Multiple miRNAs have been associated with the pathophysiology of HSCR, and serum miRNAs profiles of HSCR patients have been reported, but miRNA expression in HSCR colon tissue is almost completely unexplored. Using microarray technology, we screened colon tissue to detect miRNAs whose expression profiles were altered in HSCR and identify targets of differentially expressed miRNAs. Following filtering of low-intensity signals, data normalization, and volcano plot filtering, we identified 168 differentially expressed miRNAs (104 up-regulated and 64 down-regulated). Fifty of these mRNAs represent major targets of dysegulated miRNAs and may thus important roles in the pathophysiology of HSCR. Pathway analysis revealed that 7 of the miRNA targets encode proteins involved in regulation of cell proliferation and migration via RET and related signaling pathways (MAPK and PI3K/AKT). Our results identify miRNAs that play key roles in the pathophysiology of the complex multi-factorial disease HSCR.     

Proteomic Profiling in Multiple Sclerosis Clinical Courses Reveals Potential Biomarkers of Neurodegeneration

The aim of our project was to perform an exploratory analysis of the cerebrospinal fluid (CSF) proteomic profiles of Multiple Sclerosis (MS) patients, collected in different phases of their clinical course, in order to investigate the existence of peculiar profiles characterizing the different MS phenotypes. The study was carried out on 24 Clinically Isolated Syndrome (CIS), 16 Relapsing Remitting (RR) MS, 11 Progressive (Pr) MS patients. The CSF samples were analysed using the Matrix Assisted Laser Desorption Ionisation Time Of Flight (MALDI-TOF) mass spectrometer in linear mode geometry and in delayed extraction mode (m/z range: 1000–25000 Da). Peak lists were imported for normalization and statistical analysis. CSF data were correlated with demographic, clinical and MRI parameters. The evaluation of MALDI-TOF spectra revealed 348 peak signals with relative intensity ≥1% in the study range. The peak intensity of the signals corresponding to Secretogranin II and Protein 7B2 were significantly upregulated in RRMS patients compared to PrMS (p<0.05), whereas the signals of Fibrinogen and Fibrinopeptide A were significantly downregulated in CIS compared to PrMS patients (p<0.04). Additionally, the intensity of the Tymosin β4 peak was the only signal to be significantly discriminated between the CIS and RRMS patients (p = 0.013). Although with caution due to the relatively small size of the study populations, and considering that not all the findings remained significant after adjustment for multiple comparisons, in our opinion this mass spectrometry evaluation confirms that this technique may provide useful and important information to improve our understanding of the complex pathogenesis of MS.